This is a proposal to pursue studies on the mechanisms of relaxation of gastrointestinal smooth muscle by enteric neurotransmitters, chiefly NO and VIP, which induce relaxation of muscle tone through generation of cAMP and/or cGMP and activation of PKA and/or PKG. Our recent studies have shown: (i) that the levels of cAMP and cGMP are regulated via feedback phosphorylation of cyclases (AC V/VI and sGC) and phosphodiesterases (PDE3A, PDE4D5, and PDE5) by PKA and PKG. and (ii) that both PKA and PKG induce relaxation by inhibiting specific targets (RGS4, IP3R-I, RhoA, and MYPT1) in the signaling pathways mediating contraction. In preliminary studies, we have now identified novel targets of PKA and PKG that play equally important roles in mediating relaxation (Specific Aim I), and demonstrated the significance of scaffolding proteins, namely, AKAP79, caveolin-1, heat-shock protein (Hsp90), and PRIP-2 in modulating signaling by cAMP/PKA (Specific Aim II) and cGMP/PKG (Specific Aim III). The novel targets whose phosphorylation by PKA and/or PKG leads to relaxation include: (i) PLC-3, which mediates initial muscle contraction by Gi-coupled receptor agonists, and RGS2, which regulates Gi activity; (ii) G13 and G12/Rho-GEF (AKAP-Lbc), which regulate RhoA activity and sustained muscle contraction; and (iii) the MLC phosphatase activators, telokin and p116Rip, which promote MLC20 dephosphorylation (Specific Aim I). The strength, duration, and specificity of cAMP/PKA signaling are modulated by caveolin-1, AKAP79, and AKAP-Lbc (Specific Aim II). Preliminary studies show that caveolin-1 dampens the cAMP/PKA signal by inhibiting AC V/VI and accelerating internalization of VPAC2 receptors, whereas AKAPs dampen the cAMP/PKA signal by promoting activation of PDE4D5 and inhibition of AC V/VI; in addition, AKAP79 and AKAP-Lbc facilitate the targeting and inactivation of PLC-3, G13, Rho-GEF, and RhoA by PKA leading to muscle relaxation. The strength, duration, and specificity of cGMP/PKG signaling are modulated by caveolin-1, Hsp90, and the IP3R-I binding protein, PRIP-2 (Specific Aim III). Preliminary studies show that caveolin-1 binds PDE5 and inhibits cGMP degradation, whereas Hsp90 stabilizes sGC and enhances cGMP synthesis; PRIP-2 facilitates targeting and inactivation of IP3- RI by PKG-I. The mechanisms involving RGS2, p116Rip, PRIP-2, caveolin-1/PDE5, AKAP79/PLC-3, AKAP-Lbc/RhoA represent new discoveries resulting from our preliminary studies. The functional significance of these molecular mechanisms was confirmed in preliminary studies on muscle relaxation in integrated tissues using muscle strips and whole segments and in caveolin-1-/- mice. Analysis of these mechanisms should lead to new insights for the development of therapeutic agents that act on smooth muscle of the gut and other regions (e.g., airway and vascular smooth muscle).